Showing papers in "Emerging Infectious Diseases in 2012"

Rapid detection of carbapenemase-producing Enterobacteriaceae.

Abstract: To rapidly identify carbapenemase producers in Enterobacteriaceae, we developed the Carba NP test. The test uses isolated bacterial colonies and is based on in vitro hydrolysis of a carbapenem, imipenem. It was 100% sensitive and specific compared with molecular-based techniques. This rapid (<2 hours), inexpensive technique may be implemented in any laboratory.

Novel orthobunyavirus in Cattle, Europe, 2011.

Abstract: In 2011, an unidentified disease in cattle was reported in Germany and the Netherlands. Clinical signs included fever, decreased milk production, and diarrhea. Metagenomic analysis identified a novel orthobunyavirus, which subsequently was isolated from blood of affected animals. Surveillance was initiated to test malformed newborn animals in the affected region.

Antimicrobial drug resistance in Escherichia coli from humans and food animals, United States, 1950-2002.

Abstract: We conducted a retrospective study of Escherichia coli isolates recovered from human and food animal samples during 1950-2002 to assess historical changes in antimicrobial drug resistance. A total of 1,729 E. coli isolates (983 from humans, 323 from cattle, 138 from chickens, and 285 from pigs) were tested for susceptibility to 15 antimicrobial drugs. A significant upward trend in resistance was observed for ampicillin (p<0.001), sulfonamide (p<0.001), and tetracycline (p<0.001). Animal strains showed increased resistance to 11/15 antimicrobial agents, including ampicillin (p<0.001), sulfonamide (p<0.01), and gentamicin (p<0.001). Multidrug resistance (≥3 antimicrobial drug classes) in E. coli increased from 7.2% during the 1950s to 63.6% during the 2000s. The most frequent co-resistant phenotype observed was to tetracycline and streptomycin (29.7%), followed by tetracycline and sulfonamide (29.0%). These data describe the evolution of resistance after introduction of new antimicrobial agents into clinical medicine and help explain the range of resistance in modern E. coli isolates.

Outbreak of Porcine Epidemic Diarrhea in Suckling Piglets, China

Abstract: To the Editor: Beginning in October 2010, porcine epidemic diarrhea (PED), caused by a coronaviral infection affecting pigs, emerged in China in an outbreak characterized by high mortality rates among suckling piglets. The outbreak overwhelmed >10 provinces in southern China, and >1,000,000 piglets died. This outbreak was distinguished by ≈100% illness among piglets after birth (predominantly within 7 days and sometimes within only a few hours) and death rates of 80%–100% (Technical Appendix Table 1). Few sows or boars showed any clinical signs during the outbreak, which is not consistent with a recent report from Thailand (1). In that outbreak during late 2007, pigs of all ages were affected, exhibiting different degrees of diarrhea and no appetite. We characterized the genetic variation of the PED virus (PEDV) that caused a large-scale outbreak in China during 2010–2011 and compared it with viruses in other outbreaks. We also report a possible novel transmission pathway for PEDV. A total of 177 samples (intestine, stool, and maternal milk) were collected from pigs from different farms who had diarrhea; 100% of farms had >1 porcine sample positive for PEDV. A total of 125/177 porcine samples were confirmed as positive for PEDV by reverse transcription PCR using primers as described (2). PEDV was detected in 105 (82.0%) of 128 fecal samples and 20 (40.8%) of 49 sow milk samples. Piglets infected with PEDV showed mild hemorrhage, undigested curdled milk in the stomach, and thin-walled intestines with severe mucosal atrophy and foamy fluid (data not shown). The spike (S) gene of the family Coronaviridae has a high degree of variation and can induce neutralizing antibody (3). Reverse transcription PCR products of the 651-bp partial S gene of PEDV and the deduced amino acid sequences were aligned by using ClustalW (www.genome.jp/tools/clustalw), and a neighbor-joining tree with 1,000 bootstraps was constructed. Sequences of the S genes from this outbreak were 99.1%–100.0% homologous and had 88.7%–98.9% nt identity with all reference strains (Technical Appendix Table 2), 98.5%–98.9% with Thailand strains, and 94.5%–95.1% with vaccine strain CV777. The partial S gene deduced amino acid sequences were compared and also showed a high degree of homology (98.0%–100.0%); they had 85.3%–98.7% identity with all reference strains listed in Technical Appendix Table 2, 98.0%–98.7% with Thailand strains, and 93.3%–94.7% with vaccine strain CV777 (data not shown). Phylogenetic analysis indicated that the PEDV in the China outbreak was different from foreign and other domestic strains on the basis of the reported partial S gene sequences. All new strains were clustered in the same branch, close to the cluster of Thailand strains, and far from the cluster of vaccine strain CV777 (Figure). Figure Phylogenetic tree constructed by using the neighbor-joining method based on the 9 porcine epidemic diarrhea virus (PEDV) sequences identified in a study of porcine epidemic diarrhea in China. Partially amplified spike genes of the PEDV isolates plus 18 ... In the China outbreak, PEDV caused severe diarrheal disease in piglets; heavy economic losses in many provinces resulted, despite use of commercial vaccines (inactivated transmissible gastroenteritis [TGEV H] and porcine epidemic diarrhea [CV777]). To determine why the vaccines showed poor efficacy, we investigated evolution of the virus. Comparison of amino acid sequences from isolates from the outbreak and from the CV777 vaccine strain showed 9 amino acid mutations of fragments containing major hydrophilic regions: 16 (L→H), 18 (S→G), 22 (V→I), 44 (T→S), 89 (G→S), 100 (A→E), 107 (L→F), 130 (I→V) and 160 (I→F) (Technical Appendix Figure, panel A). Three of these 9 mutations were at positions 16, 18, and 22 in the isolates from China; they influenced the hydrophobicity of the S protein as compared with that for CV777 (Technical Appendix Figure, panel B). Phylogenic analysis showed that strain CV777 did not cluster with current common strains and showed considerable genetic distance from them. Isolates in the outbreak in China had only a minor nucleotide sequence variation from the Thailand isolates, indicating that the virus has a high genetic relatedness to the Southeast Asia strain. However, previous studies showed that isolates from Europe, South Korea, and China were serologically identical to the prototype CV777 strain (1,4). To our knowledge, fecal–oral transmission is probably the main or only route of PEDV transmission (5–7). In our study, if a fecal sample from a sick piglet was found to be positive for PEDV, we also collected and studied milk from its mother. These results showed that PEDV was present in sow milk (Technical Appendix Table 3), but the detection rate was lower for these samples (40.8%) than for the fecal samples (82.0%). On the basis of these results, we hypothesize that sow milk could represent a possible (and potentially major) route for the vertical transmission of PEDV from sow to suckling piglet. This hypothesis could be indirectly verified by our field observation that piglet death rates decreased as a result of fostering (data not shown). Our findings show that PEDV was identified not only in fecal samples from sick piglets, as expected, but also in the milk of the sow, which suggests vertical transmission of the virus.

New variants of porcine epidemic diarrhea virus, China, 2011.

Abstract: In 2011, porcine epidemic diarrhea virus (PEDV) infection rates rose substantially in vaccinated swine herds. To determine the distribution profile of PEDV outbreak strains, we sequenced the full-length spike gene from samples from 9 farms where animals exhibited severe diarrhea and mortality rates were high. Three new PEDV variants were identified.

Iatrogenic Creutzfeldt-Jakob Disease, Final Assessment

Abstract: The era of iatrogenic Creutzfeldt-Jakob disease (CJD) has nearly closed; only occasional cases with exceptionally long incubation periods are still appearing. The principal sources of these outbreaks are contaminated growth hormone (226 cases) and dura mater grafts (228 cases) derived from human cadavers with undiagnosed CJD infections; a small number of additional cases are caused by neurosurgical instrument contamination, corneal grafts, gonadotrophic hormone, and secondary infection with variant CJD transmitted by transfusion of blood products. No new sources of disease have been identified, and current practices, which combine improved recognition of potentially infected persons with new disinfection methods for fragile surgical instruments and biological products, should continue to minimize the risk for iatrogenic disease until a blood screening test for the detection of preclinical infection is validated for human use.

Hand, foot, and mouth disease caused by coxsackievirus a6.

Abstract: To the Editor: Coxsackievirus A6 (CVA6) is a human enterovirus associated with herpangina in infants. In the winter of 2012, we evaluated a cluster of 8 patients, 4 months–3 years of age, who were brought for treatment at Boston Children’s Hospital (Boston, MA, USA) with a variant of hand, foot, and mouth disease (HFMD) that has now been linked to CVA6 (Table). During this same period, the Boston Public Health Commission’s syndromic surveillance system detected a 3.3-fold increase in emergency department discharge diagnoses of HFMD. In the United States, HFMD typically occurs in the summer and early autumn and is characterized by a febrile enanthem of oral ulcers and macular or vesicular lesions on the palms and soles; the etiologic agents are most often CVA16 and enterovirus 71. Table Demographic and clinical characteristics of patients with CVA6-associated HFMD, Boston, Massachusetts, USA, 2012* In contrast to the typical manifestation, the patients in the Boston cluster exhibited symptoms in late winter (Table) and had perioral (Figure, panel A) and perirectal (Figure, panel B) papules and vesicles on the dorsal aspects of the hands and feet (Figure, panel C). Patients experienced a prodrome lasting 1–3 days, consisting of fever (8 patients), upper respiratory tract symptoms (4 patients), and irritability (7 patients). This prodrome was followed by the development of a perioral papular rash (8 patients), which was often impetiginized with secondary crusting; a prominent papulovesicular rash on the dorsum of the hands and feet (6 patients); and a perirectal eruption (7 patients). Half of the patients had intraoral lesions. Fever abated in most of the patients within a day after onset of the exanthem. The rash resolved over 7–14 days with no residual scarring. Samples from the oropharynx, rectum, and vesicles from these patients were sent to the Centers for Disease Control and Prevention (Atlanta, GA, USA) for analysis. Reverse transcription PCR and sequencing by using primers specific for a portion of the viral protein 1 coding region identified CVA6 (1) (Table). Figure Manifestations of hand, foot, and mouth disease in patients, Boston, Massachusetts, USA, 2012. Discrete superficial crusted erosions and vesicles symmetrically distributed in the perioral region (A), in the perianal region (B), and on the dorsum of the ... Outbreaks of HFMD caused by CVA6 have been described in Singapore, Finland, Taiwan, and most recently in Japan; most cases have occurred in the warmer months (2–6). Cases in the cluster described here are likely related to an emerging outbreak of CVA6-associated HFMD in the United States (7). The atypical seasonality of the outbreak, during the winter in Boston, could be related to the unusually mild temperatures in the winter of 2012. Recent CVA6 outbreaks have been characterized by a febrile illness associated with an oral enanthem and lesions on the palms, soles, and buttocks. CVA6 infections in Taiwan during 2004–2009 were associated with HFMD in 13% of cases, with disease defined as oral ulcers on the tongue or buccal mucosa and vesicular rashes on the palms, soles, knees, or buttocks (2). In Singapore, where CVA6 accounted for 24% of HFMD cases, patients had oral lesions and <5 peripheral papules, placing them on a spectrum closer to the herpangina more typically observed in CVA6 infection (8). The patients we report in this cluster most typically had perioral and perirectal papules in addition to vesicles on the dorsum of their hands. Two reports of CVA6-associated HFMD outbreaks describe cases that more closely resemble patients in the Boston outbreak. In a series from Finland in 2008, representative patients had both perioral lesions and vesicles on the dorsum of their hands (6). In a large series of patients with HFMD in Taiwan in 2010, patients with CVA6 had perioral lesions in addition to an enanthem (3). Outbreaks of CVA6-associated HFMD in Finland, Taiwan, and Japan were associated with onychomadesis, with the loss of nails occurring 1–2 months after initial symptoms (3,4,6). The association between more typical HFMD and onychomadesis has additionally been described in the United States and Europe but without a link to specific serotype or with a small percentage of CVA6-associated cases (9). Cases from the Boston epidemic may fit into an emerging clinical phenotype of CVA6, and it will be interesting to see whether nail loss develops in those patients. Given the numerous CVA6 outbreaks in multiple countries in 2008 and a US population that may be relatively naive to this serotype, CVA6 is likely to spread throughout North America. Clinicians should be aware that, although standard precautions are routinely recommended for managing enteroviral infections in health care settings, contact precautions are indicated for children in diapers to control institutional outbreaks (10). In addition, the presence of perioral lesions and peripheral vesicles on the dorsum rather than palmar/plantar surface of the hands and feet represents a unique phenotype of HFMD that could be confused with herpes simplex or varicella-zoster virus infections. Because of the atypical presentation of CVA6-associated HFMD, clinical vigilance is needed to recognize emerging regional outbreaks. More detailed epidemiologic and genetic analyses will be required to characterize the role of CVA6 in US outbreaks of HFMD.

Zika virus infection, Cambodia, 2010.

Abstract: To the Editor: Zika virus (ZIKV), a member of the family Flaviviridae, genus Flavivirus, was first isolated from the blood of a sentinel rhesus monkey from the Zika Forest of Uganda in 1948 (1). Since that time, serologic studies and virus isolations have demonstrated that the virus has a wide geographic distribution, including eastern and western Africa; the Indian subcontinent; Southeast Asia; and most recently, Micronesia (2–5). The virus is transmitted primarily through the bite of infected mosquitoes and most likely is maintained in a zoonotic cycle involving nonhuman primates (1), although recent evidence suggests the possibility of occasional sexual transmission in humans (4). Few case reports have described the clinical characteristics of ZIKV infection in humans. Most reports describe a self-limiting febrile illness that could easily be mistaken for another arboviral infection, such as dengue or chikungunya fever. We report a confirmed case of ZIKV infection in Cambodia. Since 2006, the US Naval Medical Research Unit No. 2 (NAMRU-2) has conducted surveillance for acute fever to determine causes of the infection among patients who seek health care at local clinics in Cambodia. Patients were enrolled by the health clinic physician after they gave informed consent in accordance with an institutional review board protocol approved by NAMRU-2 and the National Ethics Committee for Human Research of Cambodia. At enrollment, the physician administered a questionnaire and collected specimens (blood and throat swabs). All items were transported to the NAMRU-2 laboratory in Phnom Penh, where testing was conducted for a variety of viral, bacterial, and parasitic pathogens. In August 2010, a blood specimen was collected from a 3-year-old boy at a health clinic in Kampong Speu Province, Cambodia. The child’s reported clinical symptoms included 4 days of fever and sore throat and cough and a headache for 3 days. A maculopapular rash was not observed, and the boy was not hospitalized. The clinic staff conducted a follow-up interview and reported that the patient recovered fully. ZIKV infection was confirmed in this patient by using PCR, sequencing, and serology and through virus isolation. ELISA for chikungunya and dengue virus IgM and IgG antibodies on acute- and convalescent-phase serum was negative. A universal flavivirus real-time PCR screen that targets the nonstructural (NS) 5 gene (6) determined that the patient’s serum was positive for flavivirus RNA, but subsequent species-specific PCR ruled out 2 other flaviviruses that are highly endemic to the region (dengue and Japanese encephalitis viruses) (7–9). This result was the first nondengue, non–Japanese encephalitis virus flavivirus detected after samples from ≈10,000 enrolled patients were tested. Nucleic acid sequencing of the amplicon isolated by gel purification produced a 100-bp fragment with 100% sequence identity to ZIKV (nucleotide position 8,969 of the NS5 gene of the isolate GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU545988","term_id":"189092757"}}EU545988). ZIKV infection subsequently was serologically confirmed by hemagglutination-inhibition tests on paired serum samples. The patient’s acute-phase sample was negative, but a convalescent-phase sample gave a positive reaction with ZIKV antigen to a serum dilution of 1:320 and was negative to antigens for the 4 dengue serotypes and yellow fever and West Nile viruses. These results demonstrate that the patient had a clear monotypic flavivirus immune response with seroconversion against ZIKV, indicating a recent primary infection. The most common signs and symptoms reported in confirmed ZIKV infections are fever, headache, malaise, maculopapular rash, fatigue or myalgia, and arthritis and arthralgia (Table). In addition to fever and headache, the patient in this study had a sore throat and cough. Because of the patient’s age, additional information about symptoms was difficult to obtain. Table Reported or observed clinical signs and symptoms in persons with Zika virus infection, 1962–2010 The clinical characteristics exhibited by this case-patient are similar to those of shown in a small cluster of ZIKV infections described in Indonesia during 1977–1978 in which maculopapular rash was not observed (5). Maculopapular rash was reported as a common sign in case-patients from the recent Yap Island outbreak (3), as well as in case reports from Uganda (2), Senegal, and the United States (4), A case report of laboratory-acquired ZIKV infection also noted the lack of maculopapular rash (10). The clinical features of ZIKV infection are similar to those of dengue virus and chikungunya virus infections, and both arboviruses are found in Southeast Asia. In this region, laboratory-based confirmation is essential. The extent of ZIKV infections in Cambodia is unknown; further studies are needed to clarify the prevalence and geographic distribution of ZIKV infection in the country.

Multiple-Insecticide Resistance in Anopheles gambiae Mosquitoes, Southern Côte d’Ivoire

Abstract: Malaria control depends on mosquito susceptibility to insecticides. We tested Anopheles gambiae mosquitoes from Cote d'Ivoire for resistance and screened a subset for target site mutations. Mosquitoes were resistant to insecticides of all approved classes. Such complete resistance, which includes exceptionally strong phenotypes, presents a major threat to malaria control.

Hepatitis E virus strains in rabbits and evidence of a closely related strain in humans, France.

Abstract: Hepatitis E virus (HEV) strains from rabbits indicate that these mammals may be a reservoir for HEVs that cause infection in humans. To determine HEV prevalence in rabbits and the strains' genetic characteristics, we tested bile, liver, and additional samples from farmed and wild rabbits in France. We detected HEV RNA in 7% (14/200) of bile samples from farmed rabbits (in 2009) and in 23% (47/205) of liver samples from wild rabbits (in 2007-2010). Full-length genomic sequences indicated that all rabbit strains belonged to the same clade (nucleotide sequences 72.2%-78.2% identical to HEV genotypes 1-4). Comparison with HEV sequences of human strains and reference sequences identified a human strain closely related to rabbit strain HEV. We found a 93-nt insertion in the X domain of open reading frame 1 of the human strain and all rabbit HEV strains. These findings indicate that the host range of HEV in Europe is expanding and that zoonotic transmission of HEV from rabbits is possible.

Culicoids as vectors of Schmallenberg virus.

Abstract: To the Editor: In autumn 2011, an unidentified disease of livestock was reported on both sides of the Dutch–Germany border. By using metagenomics, the etiologic agent of this disease was identified as a novel orthobunyavirus and named Schmallenberg virus (SBV) (1). Other members of the genus Orthobunyavirus (e.g., Akabane virus) are widespread in Africa and Asia; biting midges (Culicoides spp.) and mosquitoes are responsible for transmitting these viruses. Hence, we reasonably assumed that European culicoids might be responsible for transmitting SBV within Europe. We present evidence that culicoids captured October 2011 in Denmark contained SBV RNA and most likely are vectors for this agent. In autumn 2011, culicoids were collected from several sites within Denmark. One site, a chicken farm in Hokkerup (Figure A1), was selected for study because of its location close (6 km) to the German border and proximity (<10 km) to an SBV-infected sheep farm in Germany, as reported on March 9, 2012, by the Friedrich Loeffler Institute surveillance website (www.fli.bund.de). The culicoids were collected during October 14–16 by using a Mosquito Magnet Independence trap (Mosquito Magnet, Lititz, PA, USA) baited with carbon dioxide and octenol. Midges were sorted manually into 91 specimens of the C. obsoletus group (comprising C. obsoletus, C. chiopterus, C. dewulfi, and C. scoticus) and 17 of the C. punctatus sensu stricto group, then stored at −20°C. Pools of culicoids were homogenized in water (100 µL) by using a 3-mm stainless steel bead (Dejay Distribution Ltd., Launceston, UK) in a TissueLyser II (QIAGEN, Hilden, Germany) for 1 min at 25 Hz (2). After homogenization, additional water (100 µL) was added to the samples, and then the mixture was centrifuged at 3,000 × g for 5 min. Nucleic acids were extracted from the supernatant (100 µL) by using a MagNA pure LC Total Nucleic Acid Isolation Kit on a MagNA pure LC (Roche Diagnostics, Basel, Switzerland) and eluted in water (50 µL). Two separate 1-step reverse transcription quantitative PCRs (RT-qPCRs), targeting the L segment and the S segment of SBV RNA, were performed according to protocols provided by the Friedrich Loeffler Institute in Germany (1) on the extracted nucleic acids by using a Mx3005p qPCR system (Agilent Technologies, Palo Alto, CA, USA). Another RT-qPCR targeting ruminant β-actin mRNA was performed as an internal endogenous control (3). Two of 22 pools tested strongly positive for the large (L) and small (S) segments of SBV RNA. Each positive sample was derived from 5 midges of the C. obsoletus group. One pool produced cycle threshold (Ct) values of 26.4 and 24.5 (in the L segment– and S segment–specific assays, respectively), whereas the second positive pool gave Ct values of 28.8 (L segment) and 27.6 (S segment). These pools were negative for the internal endogenous control that targeted the bovine/ovine β-actin mRNA. This result makes it unlikely that the detection of SBV RNA within the midges resulted from recent blood meals from infected animals remaining within the culicoids and suggests the virus has replicated within the midges. The PCR amplicons (145 bp; Figure) from the L segment–specific RT-qPCR were sequenced by using BigDye 1.1 chemistry on an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences of 80 bp from the amplicons, excluding the primer sequences, had 100% identity with the expected region of the SBV segment L (1). Figure RNA extracted from pools of Culicoides obsoletus group midges was tested in 1-step reverse transcription quantitative PCRs (RT-qPCRs) for the Schmallenberg virus large segment, and the products were analyzed by agarose gel electrophoresis. Lanes 1–8, ... Reported Ct values generated by using the same assays from blood of naturally infected cattle were 24–35 (1). Usually, ≈100 µL of bovine/ovine blood is used for virus detection, whereas <1 µL of blood remains in a midge after a blood meal. This uptake of blood should therefore lead to a Ct value that is at least 6–7 units higher (≈100-fold lower level of RNA) when a single midge is tested by RT-qPCR (4). Thus, even if all 5 culicoids in a pool had recently taken a blood meal from a viremic animal, the Ct values observed here strongly suggest replication of SBV within the C. obsoletus group midges. However, in principle, other hosts of SBV could have a much higher level of viremia than cattle and could provide the levels of SBV RNA detected. C. punctatus s.s. midges cannot be ruled out as a possible vector of SBV because of the limited number of insects tested. Our study demonstrates the presence of SBV RNA in C. obsoletus group midges caught in Denmark during October 2011. The low Ct values (i.e., high SBV RNA levels) and the absence of ruminant β-actin mRNA in these samples strongly suggest that SBV replicates in these midges and hence that the C. obsoletus group midges are natural vectors for this virus.

Epidemiology of foodborne norovirus outbreaks, United States, 2001-2008.

Abstract: Noroviruses are the leading cause of foodborne illness in the United States. To better guide interventions, we analyzed 2,922 foodborne disease outbreaks for which norovirus was the suspected or confirmed cause, which had been reported to the Foodborne Disease Outbreak Surveillance System of the Centers for Disease Control and Prevention during 2001-2008. On average, 365 foodborne norovirus outbreaks were reported annually, resulting in an estimated 10,324 illnesses, 1,247 health care provider visits, 156 hospitalizations, and 1 death. In 364 outbreaks attributed to a single commodity, leafy vegetables (33%), fruits/nuts (16%), and mollusks (13%) were implicated most commonly. Infected food handlers were the source of 53% of outbreaks and may have contributed to 82% of outbreaks. Most foods were likely contaminated during preparation and service, except for mollusks, and occasionally, produce was contaminated during production and processing. Interventions to reduce the frequency of foodborne norovirus outbreaks should focus on food workers and production of produce and shellfish.

Characterization of Mycobacterium orygis as M. tuberculosis complex subspecies.

Abstract: The oryx bacilli are Mycobacterium tuberculosis complex organisms for which phylogenetic position and host range are unsettled. We characterized 22 isolates by molecular methods and propose elevation to subspecies status as M. orygis. M. orygis is a causative agent of tuberculosis in animals and humans from Africa and South Asia.

Control of Fluoroquinolone Resistance through Successful Regulation, Australia

Abstract: Fluoroquinolone antimicrobial drugs are highly bioavailable, broad-spectrum agents with activity against gram-negative pathogens, especially those resistant to other classes of antimicrobial drugs. Australia has restricted the use of quinolones in humans through its national pharmaceutical subsidy scheme; and, through regulation, has not permitted the use of quinolones in food-producing animals. As a consequence, resistance to fluoroquinolones in the community has been slow to emerge and has remained at low levels in key pathogens, such as Escherichia coli. In contrast to policies in most other countries, this policy has successfully preserved the utility of this class of antimicrobial drugs for treatment of most infections.

Hand, foot, and mouth disease caused by coxsackievirus A6, Japan, 2011.

Abstract: To the Editor: Coxsackievirus A6 (CVA6) belongs to human enterovirus species A of the genus Enterovirus. According to a Japanese Infectious Agents Surveillance Report, this virus is one of the major causes of herpangina, an acute febrile disease characterized by vesicles, ulcers, and redness around the uvula, which occurs mainly in young children and infants (1). In June 2011, a sudden increase in cases of hand, foot, and mouth disease (HFMD) at pediatric sentinel sites (≈3,000 pediatric hospitals and clinics) was reported to the National Epidemiologic Surveillance of Infectious Diseases System in Japan. Compared with past numbers of cases over 30 years of surveillance, the number of cases of HFMD per sentinel site peaked in week 28 (July) of 2011 (10.97 cases per sentinel), particularly in western Japan (2). According to the Infectious Agents Surveillance Report (as of September, 18, 2011), CVA6 was detected in 709 HFMD cases and 156 herpangina cases throughout Japan (1). Clinical samples (throat swab specimens and feces) obtained from sentinel sites in Shimane, Hyogo, Hiroshima, and Shizuoka, Japan, were screened for enteroviruses by using an enterovirus-specific reverse transcription PCR and sequence analysis of the partial viral protein (VP)4/VP2 or VP1 region (3). Among 93 clinical samples from 108 HFMD case-patients, we identified 74 case-patients as CVA6 positive by sequence analysis. On the basis of sequence analysis of the entire VP1 region (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"AB649286-AB649291","start_term":"AB649286","end_term":"AB649291","start_term_id":"342166534","end_term_id":"342166544"}}AB649286-AB649291), the consensus sequence had 82.3%–82.5% nt identity (94.8%–95.4% aa identity) with the prototype CVA6 Gdula strain (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY421764","term_id":"40068436"}}AY421764). CVA6 was not isolated from clinical samples in a cell culture system. Therefore, most CVA6 strains were identified by molecular detection directly from clinical samples and sequence analysis. Some CVA6 strains were grown and isolated in suckling mice; these strains were antigenically identified as CVA6 by a neutralization test with specific antiserum against CVA6 (4). In Japan, HFMD and herpangina are classified as category V infectious diseases. On the basis of clinical diagnosis, suspected infections were reported by pediatric sentinel sites on a weekly basis to the Infectious Disease Surveillance Center of the National Institute of Infectious Diseases (Tokyo, Japan). Typical clinical signs and symptoms of HFMD cases caused by CVA6 were fever, mild vesicles in oral mucosa, and skin blisters on hands, arms, feet, legs, buttocks, and nail matrixes (Figure). Some patients with HFMD had onychomadesis (periodic shedding of the nails) 1–2 months after onset of HFMD. Most cases of HFMD were self-limited. However, additional follow-up may be necessary for patients with onychomadesis who are treated at dermatology clinics. Figure Typical clinical manifestations of hand, foot, and mouth disease associated with coxsackievirus CVA6 in Shizuoka, Japan, June–July, 2011. A) Hand and arm of a 2.5-year-old boy; B) foot and C) buttocks of a 6-year-old boy; D) nail matrix of a 20-month ... As in other countries in the Asia–Pacific region, major causes of HFMD in Japan were CVA16 and enterovirus 71. In 2010, enterovirus 71 was identified as a major cause of HFMD (2). In contrast, CVA6 was consistently associated with herpangina, as were CVA2, CVA4, CVA5, and CVA10, but CVA6 was occasionally detected in HFMD case-patients. CVA6 was the major cause of herpangina in 2007, but an increase in the detection rate of CVA6 in HFMD case-patients was reported in Japan in 2009 (2). HFMD outbreaks caused by CVA6 were reported in Singapore, Finland, and Taiwan in 2007–2009 (5–8). Recent HFMD outbreaks in Finland and Spain were associated with cases of onychomadesis 1–2 months after onset of HFMD (6,8,9). In Japan, cases of onychomadesis after onset of HFMD were reported in 2009 (10). Therefore, changes in clinical outcomes of CVA6-associated diseases should be investigated. Although most HFMD cases caused by CVA6 in Japan were mild, CVA6 was also detected in other clinical samples, including cerebrospinal fluid from a patient with acute encephalitis in Hiroshima, which reaffirmed possible additional clinical manifestations during an HFMD outbreak caused by CVA6. Careful surveillance of disease and infectious agent activities are crucial in monitoring CVA6-associated HFMD, onychomadesis, and neurologic diseases. Nucleotide identity between CVA6 strains in Finland (2008) (7) and Japan (2011) was ≈95% in the partial VP1 region. More detailed genetic, phenotypic, and epidemiologic analyses of CVA6 are needed to determine the role of CVA6 in HFMD outbreaks with or without onychomadesis.

Nonpasteurized dairy products, disease outbreaks, and state laws-United States, 1993-2006.

Abstract: Although pasteurization eliminates pathogens and consumption of nonpasteurized dairy products is uncommon, dairy-associated disease outbreaks continue to occur. To determine the association of outbreaks caused by nonpasteurized dairy products with state laws regarding sale of these products, we reviewed dairy-associated outbreaks during 1993-2006. We found 121 outbreaks for which the product's pasteurization status was known; among these, 73 (60%) involved nonpasteurized products and resulted in 1,571 cases, 202 hospitalizations, and 2 deaths. A total of 55 (75%) outbreaks occurred in 21 states that permitted sale of nonpasteurized products; incidence of nonpasteurized product-associated outbreaks was higher in these states. Nonpasteurized products caused a disproportionate number (≈150× greater/unit of product consumed) of outbreaks and outbreak-associated illnesses and also disproportionately affected persons <20 years of age. States that restricted sale of nonpasteurized products had fewer outbreaks and illnesses; stronger restrictions and enforcement should be considered.

Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis.

Abstract: A direct rapid immunohistochemical test (dRIT) was evaluated under field and laboratory conditions to detect rabies virus antigen in frozen and glycerol-preserved field brain samples from northwestern Tanzania. Compared to the direct fluorescent antibody test, the traditional standard in rabies diagnosis, the dRIT was 100% sensitive and specific.

Workshop on treatment of and postexposure prophylaxis for Burkholderia pseudomallei and B. mallei Infection, 2010.

Abstract: The US Public Health Emergency Medical Countermeasures Enterprise convened subject matter experts at the 2010 HHS Burkholderia Workshop to develop consensus recommendations for postexposure prophylaxis against and treatment for Burkholderia pseudomallei and B. mallei infections, which cause melioidosis and glanders, respectively. Drugs recommended by consensus of the participants are ceftazidime or meropenem for initial intensive therapy, and trimethoprim/sulfamethoxazole or amoxicillin/clavulanic acid for eradication therapy. For postexposure prophylaxis, recommended drugs are trimethoprim/sulfamethoxazole or co-amoxiclav. To improve the timely diagnosis of melioidosis and glanders, further development and wide distribution of rapid diagnostic assays were also recommended. Standardized animal models and B. pseudomallei strains are needed for further development of therapeutic options. Training for laboratory technicians and physicians would facilitate better diagnosis and treatment options.

Variant rabbit hemorrhagic disease virus in young rabbits, Spain.

Abstract: Outbreaks of rabbit hemorrhagic disease have occurred recently in young rabbits on farms on the Iberian Peninsula where rabbits were previously vaccinated. Investigation identified a rabbit hemorrhagic disease virus variant genetically related to apathogenic rabbit caliciviruses. Improved antivirus strategies are needed to slow the spread of this pathogen.

Novel Hepatitis E Virus in Ferrets, the Netherlands

Abstract: To the Editor: Hepatitis E virus (HEV), a member of the family Hepeviridae and the genus Hepevirus, is transmitted by the fecal–oral route and causes liver inflammation, which leads to mortality rates of ≤20% in pregnant woman (1,2). Human hepatitis E is a major disease not only in developing countries but also in industrialized countries, and identification of animal strains of HEV in pigs and deer and its zoonotic potential has raised considerable public health concerns (1,3). Recent reports suggest that other animals such as rats, mongooses, chickens, rabbits, and trout also may harbor HEVs (1–5). The genomes of these viruses are ≈6.6 kb–7.2 kb and encode 3 open reading frames (ORFs) flanked by a capped 5′ end and a poly A tail at the 3′ end (1,3). We used random PCR amplification and high-throughput sequencing technology to investigate HEV sequences in ferrets (Mustela putorius) from the Netherlands. In 2010, fecal samples were collected from ferrets in the Netherlands and stored at −80°C. Samples that were negative for ferret coronavirus (6) were further characterized for other pathogens. Viral RNA was isolated and viral metagenomic libraries were constructed for 454 pyrosequencing as described (7,8), and 248,840 sequence reads were generated from 7 fecal samples. Using Blastn and Blastx (www.ncbi.nlm.nih.gov/BLAST), we identified 289 sequence reads in 1 sample that were related to rat HEV and that could be assembled into 6 contigs covering ≈50% of the ferret HEV (FRHEV) genome. We then developed a set of nested PCR primers on the basis of obtained sequences to detect viral RNA (Technical Appendix Table 1). Total RNA extracted from 43 ferret fecal samples collected from 19 locations in the Netherlands was used to perform reverse transcription PCR amplification. Using this PCR, we detected viral RNA in 4 (9.3%) fecal samples tested from 4 locations (distance between each sampling location ranged from 25 km to 127 km). All amplicons were confirmed by nucleotide sequencing. We have limited information regarding the clinical disease this virus may cause because these samples were obtained from household pet ferrets that did not show overt clinical signs. In addition, 4/16 animals from a single farm were IgG positive when tested for IgG against HEV by using recombinant human HEV protein (Wantai, Beijing, China). To characterize the complete genome of this virus, we selected 2 PCR-positive samples (FRHEV4 and FRHEV20), developed different sets of specific primers on the basis of sequence fragments obtained by 454 pyrosequencing, directly sequenced amplicons by Sanger sequencing, and used a rapid amplification of cDNA ends PCR to obtain 5′ and 3′ frame end sequences. Using overlapping fragments we assembled 2 complete FRHEV genome sequences that contained 6,854 nt, including a 13-nt 3′ poly A tail and a 12-nt 5′ end. FRHEV full-genome sequences FRHEV4 and FRHEV20 showed 98.6% sequence identity and were deposited into GenBank under accession nos. {"type":"entrez-nucleotide","attrs":{"text":"JN998606","term_id":"397310723","term_text":"JN998606"}}JN998606 and {"type":"entrez-nucleotide","attrs":{"text":"JN998607","term_id":"397310728","term_text":"JN998607"}}JN998607, respectively. The FRHEV genome contains a complete ORF1 gene that encodes a nonstructural protein of 1,596 aa, an ORF2 gene that encodes a capsid protein of 654 aa, an ORF3 gene that encodes a phosphoprotein of 108 aa, and a 3′ noncoding region of 78 nt. Sequence analyses indicated that the FRHEV genome shared the highest identity (72.3%) with rat HEV. Sequence identity with HEV genotypes 1–4 and rabbit and avian HEVs ranged from 54.5% to 60.5% (Technical Appendix Table 2). The FRHEV genome organization was found to be slightly different from other HEVs and included a putative ORF (ORF4) of 552 nt that overlapped with ORF1 (Technical Appendix Figure). A similar pattern of genome organization was observed for both FRHEVs. Phylogenetic analysis of the complete genomes clearly showed that FRHEV was separated from genotype 1–4 HEVs and clustered with rat HEV (Figure). Similar phylogenetic clustering was observed when nucleotide and deduced amino acid sequences of ORF1, ORF2, and ORF3 were analyzed separately. The phylogenetic distance between rat HEV and FRHEV is larger than the distance between genotype 1 and genotype 2 HEV. Figure Phylogenetic tree based on the complete genomic sequences of ferret hepatitis E viruses (HEVs) and human, rabbit, swine, avian, and rat HEV strains. Names of HEV strains follow GenBank accession numbers. Sequence alignment was performed by using ClustalW ... In recent years, an increasing number of sporadic cases of hepatitis E have been reported (1,9). Several observations suggest that autochthonous cases are caused by zoonotic spread of infection from wild or domestic animals (1,3,9). In addition, IgG anti-HEV seropositivity in the United States has been associated with several factors, including having a pet at home (10). Further studies are needed to identify the zoonotic potential of FRHEV. Technical Appendix: Genome organization of hepatitis E viruses (HEVs) and initiation of translation of open reading frame 1 (ORF1), ORF2, and ORF3 of ferret HEV. Click here to view.(190K, pdf)

Real-time estimates in early detection of SARS

Abstract: We propose a Bayesian statistical framework for estimating the reproduction number R early in an epidemic. This method allows for the yet-unrecorded secondary cases if the estimate is obtained before the epidemic has ended. We applied our approach to the severe acute respiratory syndrome (SARS) epidemic that started in February 2003 in Hong Kong. Temporal patterns of R estimated after 5, 10, and 20 days were similar. Ninety-five percent credible intervals narrowed when more data were available but stabilized after 10 days. Using simulation studies of SARS-like outbreaks, we have shown that the method may be used for early monitoring of the effect of control measures.

Occurrence, Transmission, and Zoonotic Potential of Chronic Wasting Disease

Abstract: Chronic wasting disease (CWD) is a fatal, transmissible prion disease that affects captive and free-ranging deer, elk, and moose. Although the zoonotic potential of CWD is considered low, identification of multiple CWD strains and the potential for agent evolution upon serial passage hinders a definitive conclusion. Surveillance for CWD in free-ranging populations has documented a continual geographic spread of the disease throughout North America. CWD prions are shed from clinically and preclinically affected hosts, and CWD transmission is mediated at least in part by the environment, perhaps by soil. Much remains unknown, including the sites and mechanisms of prion uptake in the naive host. There are no therapeutics or effective eradication measures for CWD-endemic populations. Continued surveillance and research of CWD and its effects on cervid ecosystems is vital for controlling the long-term consequences of this emerging disease.

Crimean-Congo hemorrhagic fever virus in ticks, Southwestern Europe, 2010.

Abstract: To the Editor: Crimean-Congo hemorrhagic fever virus (CCHFV; family Bunyaviridae, genus Nairovirus) causes outbreaks of severe hemorrhagic fever in humans, with case-fatality rates <30% (1,2). The disease was initially recognized by Russian scientists in the 1940s (3), and the virus was first isolated in the Democratic Republic of Congo some years later (4). CCHFV is reported throughout broad regions of Africa, Europe, the Middle East, and Asia. Reports linking transmission of the virus with an infected vector have involved ticks of the genus Hyalomma (5). It appears that maintenance of active foci of CCHFV in the field is dependent on Hyalomma spp., even within periods of silent activity. Several vertebrates are involved in the natural transmission cycle (6). Transmission of CCHFV to humans occurs through tick bites, direct contact with blood or tissues of infected animals, person-to-person spread, or by nosocomial infection (1). In southeastern Europe, the Balkans are the known western limit for CCHFV (7). This finding is of special interest because Hyalomma marginatum, the main tick vector in the western Paleartic (an ecozone that includes temperate and cold areas of Eurasia and North Africa and several archipelagos and islands in the Atlantic and Pacific Oceans), is common throughout the Mediterranean Basin (7), where clinical cases of the disease or the virus have not been reported. Unsupported claims of the effects of climate on virus distribution have been reported but never empirically demonstrated (8). We report the detection of CCHFV in ticks collected in southwestern Europe. A total of 117 semi-engorged adult H. lusitanicum ticks were collected from 28 adult red deer (Cervus elaphus) in November 2010, at a site (39.63°N, 7.33°W) in Caceres, Spain. Live ticks were transported to the special pathogens laboratory at Hospital San Pedro–CIBIR in Logrono (northern Spain), classified, and frozen at −80°C. For RNA extraction, specimens were washed in 70% ethanol and then in Milli-Q water (Milli-Q Advantage water system; Millipore Iberica, S.A., Madrid, Spain) that had been autoclaved. Each tick was cut lengthwise; half was used for additional processing and the remainder was stored. Before use, each half was crushed in sterile conditions. RNA was individually extracted by using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and frozen at −80°C. The RNA was distributed in 12 pools and retrotranscribed by using the Omniscript RT kit (QIAGEN) according to the manufacturer’s instructions and then frozen at −20°C. Nested PCRs were performed by using specific primers for the small segment of CCHFV as described (9). Negative controls (with template DNA but without primers and with primers and containing water instead of template DNA) were included in all assays. For the second round of PCRs, 2 of 12 pools showed amplicons of the expected size (211 bp). Only 1 amplicon could be sequenced. MEGA5 (www.megasoftware.net) was used to compare the sequence with representative small segment sequences of CCHFV available in GenBank (Figure). (Aligned sequences are available from the authors.) Pools of cDNA were submitted to the Spanish National Center of Microbiology (Madrid), where results were confirmed. The CCHFV sequence we report showed 98% genetic similarity (204/209 bp) with sequences recorded for CCHFV in Mauritania and Senegal, on the western coast of Africa. Figure Evolutionary relationships of Crimean-Congo hemorrhagic fever virus strains from Spain and other representative sites. Evolutionary history was inferred by using the unweighted pair group method with arithmetic mean. The optimal tree is shown (sum of ... This finding suggests the circulation of CCHFV in southwestern Europe. The close affinity of the strain from Spain with strains circulating in western Africa and the lack of similarity with isolates from eastern Europe suggest the introduction of this virus from nearby countries of northern Africa. Migratory movements of birds could explain the presence of the virus in southwestern Europe because birds are common hosts of immature H. marginatum, which was reportedly introduced into Europe through annual migratory flights along the western coast of Africa (10). Because of the lack of genetic similarities among virus strains, trade movements of domestic or wild ungulates from eastern Europe do not support our finding. We cannot state whether this virus was circulating previously or if other strains are present in the area because CCHFV detection in the western Mediterranean region has not been previously addressed. H. lusitanicum ticks exist as relatively isolated populations in a narrow strip from Sicily to Portugal. The Mediterranean rabbit and ungulates, the main hosts for immature and adult H. lusitanicum ticks, respectively, are residents of the collection area; however, the movement of these animals through trade has not occurred for several years. Thus, H. lusitanicum ticks could not serve as a spreading vector in the western Mediterranean region. The CCHFV strain from southwestern Europe has been found in ticks restricted to hosts that cannot spread long distances. Therefore, although it would be unlikely, given the strain’s similarity with CCHFV isolates from Senegal and Mauritania, we should not exclude the possibility of an ancient existence for this strain. Additional data collected in the Mediterranean Basin are necessary to establish the actual range of CCHFV.

Chicken as Reservoir for Extraintestinal Pathogenic Escherichia coli in Humans, Canada

Abstract: We previously described how retail meat, particularly chicken, might be a reservoir for extraintestinal pathogenic Escherichia coli (ExPEC) causing urinary tract infections (UTIs) in humans. To rule out retail beef and pork as potential reservoirs, we tested 320 additional E. coli isolates from these meats. Isolates from beef and pork were significantly less likely than those from chicken to be genetically related to isolates from humans with UTIs. We then tested whether the reservoir for ExPEC in humans could be food animals themselves by comparing geographically and temporally matched E. coli isolates from 475 humans with UTIs and from cecal contents of 349 slaughtered animals. We found genetic similarities between E. coli from animals in abattoirs, principally chickens, and ExPEC causing UTIs in humans. ExPEC transmission from food animals could be responsible for human infections, and chickens are the most probable reservoir.

Pandemic influenza threat and preparedness.

Abstract: The threat of a human influenza pandemic has greatly increased over the past several years with the emergence of highly virulent avian influenza viruses, notably H5N1 viruses, which have infected humans in several Asian and European countries. Previous influenza pandemics have arrived with little or no warning, but the current widespread circulation of H5N1 viruses among avian populations and their potential for increased transmission to humans and other mammalian species may afford us an unprecedented opportunity to prepare for the next pandemic threat. The US Department of Health and Human Services is coordinating a national strategy to respond to an influenza pandemic that involves multiple agencies, including the Centers for Disease Control and Prevention, the Food and Drug Administration, and the National Institutes of Health (NIH). Within NIH, the National Institute of Allergy and Infectious Diseases (NIAID) conducts basic and clinical research to develop new vaccine technologies and antiviral drugs against influenza viruses. We describe recent research progress in preparing for pandemic influenza.

Trichomonas vaginalis antimicrobial drug resistance in 6 US cities, STD Surveillance Network, 2009-2010.

Abstract: Nitroimidazoles (metronidazole and tinidazole) are the only recommended drugs for treating Trichomonas vaginalis infection, and previous samples that assessed resistance of such isolates have been limited in geographic scope. We assessed the prevalence of in vitro aerobic metronidazole and tinidazole resistance among T. vaginalis isolates from multiple geographic sites in the United States. Swab specimens were obtained from women who underwent routine pelvic examinations at sexually transmitted disease clinics in 6 US cities. Cultured T. vaginalis isolates were tested for nitroimidazole resistance (aerobic minimum lethal concentration [MLC] >50 µg/mL). Of 538 T. vaginalis isolates, 23 (4.3%) exhibited low-level in vitro metronidazole resistance (minimum lethal concentrations 50–100 µg/mL). No isolates exhibited moderate- to high-level metronidazole resistance or tinidazole resistance. Results highlight the possibility that reliance on a single class of antimicrobial drugs for treating T. vaginalis infections may heighten vulnerability to emergence of resistance. Thus, novel treatment options are needed.

Hepatitis E virus seroprevalence among adults, Germany.

Abstract: We assessed hepatitis E virus (HEV) antibody seroprevalence in a sample of the adult population in Germany. Overall HEV IgG prevalence was 16.8% (95% CI 15.6%–17.9%) and increased with age, leveling off at >60 years of age. HEV is endemic in Germany, and the lifetime risk for exposure is high.

Spike Protein Fusion Peptide and Feline Coronavirus Virulence

Abstract: Coronaviruses are well known for their potential to change their host or tissue tropism, resulting in unpredictable new diseases and changes in pathogenicity; severe acute respiratory syndrome and feline coronaviruses, respectively, are the most recognized examples. Feline coronaviruses occur as 2 pathotypes: nonvirulent feline enteric coronaviruses (FECVs), which replicate in intestinal epithelium cells, and lethal feline infectious peritonitis viruses (FIPVs), which replicate in macrophages. Evidence indicates that FIPV originates from FECV by mutation, but consistent distinguishing differences have not been established. We sequenced the full genome of 11 viruses of each pathotype and then focused on the single most distinctive site by additionally sequencing hundreds of viruses in that region. As a result, we identified 2 alternative amino acid differences in the putative fusion peptide of the spike protein that together distinguish FIPV from FECV in >95% of cases. By these and perhaps other mutations, the virus apparently acquires its macrophage tropism and spreads systemically.

Outbreak of extended-spectrum β-lactamase-producing Klebsiella oxytoca infections associated with contaminated handwashing sinks(1).

Abstract: Klebsiella oxytoca is primarily a health care-associated pathogen acquired from environmental sources. During October 2006-March 2011, a total of 66 patients in a hospital in Toronto, Ontario, Canada, acquired class A extended-spectrum β-lactamase-producing K. oxytoca with 1 of 2 related pulsed-field gel electrophoresis patterns. New cases continued to occur despite reinforcement of infection control practices, prevalence screening, and contact precautions for colonized/infected patients. Cultures from handwashing sinks in the intensive care unit yielded K. oxytoca with identical pulsed-field gel electrophoresis patterns to cultures from the clinical cases. No infections occurred after implementation of sink cleaning 3×/day, sink drain modifications, and an antimicrobial stewardship program. In contrast, a cluster of 4 patients infected with K. oxytoca in a geographically distant medical ward without contaminated sinks was contained with implementation of active screening and contact precautions. Sinks should be considered potential reservoirs for clusters of infection caused by K. oxytoca.

Loss of Household Protection from Use of Insecticide-Treated Nets against Pyrethroid-Resistant Mosquitoes, Benin

Abstract: Pyrethroid resistance is becoming widespread in Anopheles gambiae mosquitoes, coinciding with expanded use of insecticide-treated nets (ITNs) throughout Africa. To investigate whether nets in use are still protective, we conducted household trials in northern and southern Benin, where An. gambiae mosquitoes are susceptible and resistant, respectively, to pyrethroids. Rooms were fi tted with window traps and monitored for mosquito biting and survival rates before and after the nets were treated with pyrethroid. Sleeping under an ITN in the location with resistant mosquitoes was no more protective than sleeping under an untreated net, regardless of its physical condition. By contrast, sleeping under an ITN in the location with susceptible mosquitoes decreased the odds of biting by 66%. ITNs provide little or no protection once the mosquitoes become resistant and the netting acquires holes. Resistance seriously threatens malaria control strategies based on ITN.